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. 2019 Nov 9;22:453–465. doi: 10.1016/j.isci.2019.11.013

Figure 2.

Figure 2

Gephyrin Protein Levels at Synapses Are Not Altered by Forskolin

(A) Sample images showing the effect of forskolin treatment on GlyRα1 (magenta in the merged image) and two different gephyrin antibodies (mAb7a and 3B11, green). Fluorescent signals from Geph 7a were reduced by forskolin, whereas GlyRα1 and Geph 3B11 were not affected. Scale bar, 5 μm.

(B) Integrated fluorescence intensity of endogenous gephyrin at rat spinal cord synapses was quantified in GlyRα1-positive puncta (background-corrected data). Geph 7a labeling was decreased by 45% ± 4% after 30-min treatment with 20 μM forskolin (For) compared with the control (***p < 0.001, ANOVA), whereas no significant changes (NS) were observed with Geph 3B11 (GlyRα1: nCtr = 149, nFor = 144 cells, 5 experiments; Geph 7a: nCtr = 60, nFor = 56, 2 experiments; Geph 3B11: nCtr = 89, nFor = 88 cells, 3 experiments). Data are represented as 10%, 25%, 50%, 75%, and 90% percentiles; the mean is indicated as a cross.

(C) Dissociated spinal cord cultures prepared from an mRFP-gephyrin knock-in mouse strain (mRFP-Geph KI, magenta) were treated with or without forskolin and then immunolabeled for GlyRα1 (green). Scale bar, 5 μm.

(D) No significant change in mRFP-Geph KI fluorescence and GlyRα1 immunoreactivity was observed (nCtr = 39; nFor = 40 cells, 1 experiment).

(E) Organotypic spinal cord cultures made from mRFP-gephyrin KI mice were treated at DIV21 with 20 μM forskolin for 60 min and fixed, labeled, and analyzed by confocal microscopy. Geph 7a immunoreactivity (green in merged image) and mRFP fluorescence (magenta) were quantified in a consecutive series of nine single-plane images taken from the dorsal edge of the slice toward the center (nCtr = 44, nFor = 57 images from 7–8 slices and 2 independent experiments; ***p < 0.0001 for the pooled data points for each condition, t test; data are shown as mean ± SD). The sample images represent a non-treated control slice; the gray outline illustrates the shape of the complete organotypic slice. Scale bar, 100 μm; zoomed images, 5 μm.